In vivo induction for enhanced function of isolated hepatocytes

ABSTRACT

The invention features a liver cell culture comprising hepatocytes that have increased detoxification enzyme activity when isolated from a liver of a donor that had been administered at least one induction agent prior isolation of hepatocyte cells. The induced hepatocytes are used in a bioreactor and cultured to produce hepatocyte cell products or metabolize toxins added to the culture. The bioreactor is, or is an integral part of, a liver assist device used to treat a patient in need of liver assist.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a divisional of co-pending application Ser. No.09/621,921, filed Jul. 24, 2000, which claims the benefit of provisionalapplication Serial No. 60/145,362, filed Jul. 22, 1999.

FIELD OF THE INVENTION

[0002] The field of the invention is cell culture and medicalbiotechnology, particularly hepatocyte cell cultures used in liverassist devices for treating a patient with liver disease. Hepatocytecells are induced in vivo, procured from the liver organ, cultured andincorporated in a device to treat a patient via the bloodstream toprovide hepatic function. The hepatocyte isolation methods of theinvention provide enhanced cell function that extends the functionalityof the cells in the course of treating the patient.

BACKGROUND OF THE INVENTION

[0003] Extracorporeal liver assist devices (LAD) have been proposed as atreatment for patients in acute or fulminant liver failure. The LADwould function as a temporary support designed to provide hepaticfunction until liver transplantation or the regeneration of thepatient's own liver. The LAD incorporates a bioreactor containingisolated porcine hepatocytes that are expected to detoxify substances inthe circulating plasma of patients in liver failure. However, one of thechallenges in using isolated hepatocytes is that many of thesedifferentiated activities are transient, lasting only hours to a fewdays in culture (Nishibe, Y, and Hirata, M. Induction of cytochromeP-450 isozymes in cultured monkey hepatocytes. Int J Biochem Cell Bio.27:3:279-285. 1995. Jauregui, H O, Ng, S F, Gann, K L and Waxman, D J.Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) andassociated monooxygenase activities in primary cultures of adult rathepatocytes. Xeno, 21(9):1091-106. 1991. Niak, S, Trenkler, D,Santangini, H, Pan, J and Jauregui, H O. Isolation and culture ofporcine hepatocyte for artificial liver support. Cell Trans 5:107-115,1996.) These functional detoxification activities exist as a family ofenzymes, including cytochrome P450 isoenzymes, with each enzymeresponsible for metabolism of specific substrates.

[0004] While the roles of hepatocytes in a LAD are multifold, one oftheir most critical functions is detoxification mediated bydetoxification enzymes. Therefore, the maintenance of P450 cytochromeand other detoxification activity of hepatocyte cultures is of interestin the successful treatment of fulminant hepatic failure with a liverassist device.

[0005] The method of the invention increases enzyme activity in normalhepatocytes as much as 100-fold or more and that enhanced activity ismaintained for at least one week in culture. This sustained level ofdetoxification activity from in vivo induction methods is significantlyhigher than levels found in non-induced hepatocytes or those obtainedusing in vitro induction methods. The cells maintain this level offunction when incorporated in bioreactor culture to produce cellproducts and metabolize toxic substances. The invention described herewould serve the medical community by increasing the detoxificationcapabilities of hepatocytes to be used therapeutically when thebioreactor is used as, or incorporated into, a liver assist device.

SUMMARY OF THE INVENTION

[0006] The invention features a liver cell culture comprisinghepatocytes that have increased functional enzyme activity when isolatedfrom a liver of a donor that had been administered at least oneinduction agent in vivo prior to isolation of hepatocyte cells from theliver. The induced hepatocytes are used in a bioreactor and cultured toproduce hepatocyte cell products or metabolize toxins added to theculture, or both. In the preferred embodiment, the bioreactor is, or isan integral part of, a liver assist device used to treat a patient inneed of liver assist. In another preferred embodiment at least twocultures of hepatocytes from different isolations induced by differentinduction agents may be mixed or used together in a bioreactor to have abioreactor that exhibits a wider range of increased functional enzymeactivity.

DESCRIPTION OF THE FIGURES

[0007]FIG. 1 presents the effects of phenobarbital on in vitro and invivo induced hepatocytes. In vitro induction of CYPIIB2 (BROD), FIG. 1a,and CYPIIB1 (PROD), FIG. 1b, with phenobarbital (PB) is shown. In vivoinduction of CYPIIB2 (BROD), FIG. 1c, and CYPIB1 (PROD), FIG. 1d, isalso shown.

[0008]FIG. 2 shows maintenance of function of CYPIIB2 and CYPIIB1isozymes at four days after plating of hepatocytes induced withphenobarbital (PB) in vivo and in vitro.

[0009]FIG. 3 depicts the effects of 3-methylcholanthrene (“3 MC” or“MC”) on both in vitro and in vivo induced hepatocyte cultures. In vitroinduction of CYPIA2 (MROD) is shown in FIG. 3a, and CYPIA1 (EROD) inFIG. 3b. FIGS. 3c, d, and e demonstrate the impact that3-methylcholanthrene possessed in vivo for CYPIIB1 (PROD), CYPIA2(MROD), and CYPIA1 (EROD), respectively.

[0010]FIG. 4 shows that 7-ethoxycoumarin O-deethylation is higher whenhepatocytes are induced in vivo with phenobarbital than noninducedcontrol cultures.

DETAILED DESCRIPTION OF THE INVENTION

[0011] Heretofore, cell cultures from liver procured from induced donorshave not been incorporated in a bioreactor, particularly for use in aliver assist device.

[0012] In the method to obtain induced cells, a liver donor is selectedand screened for appropriate age and health necessary to obtain healthycells from the donor's organs. The liver donor for obtaining hepatocytesis preferably a normal or transgenic animal donor of either mammalian orrodent species, more preferably of equine, canine, porcine, bovine,ovine, or murine species; and most preferably, a porcine donor. Due tothe ease of handling smaller animals and liver organs, pigs betweenabout 5 kg to about 20 kg are used, preferably about 8 kg, but any sizedonor may be used as a source for liver organs.

[0013] Induction is preferably performed by administering at least oneinduction agent to an animal donor via direct injection to thebloodstream, intraperitoneally, or intramuscularly; however, inductionagents may also be administered to a donor using other routes such asorally, transdermally, or by inhalation. One or more agents may beadministered at one time in a single dose or over a time as separateddoses of different induction agents. The donor may be dosed with acombination of two or more induction agents to upregulate certaindesired detoxification enzymes to create a hepatocyte culture having acustomized enzyme activity profile. The dosing of the induction agentmay be administered in a single day or over a time, such as over anumber of hours or days, before isolating the hepatocyte cells from thedonor liver. For example, some induction agents such as phenobarbitalare relatively unstable molecules after injection to a donor and are,therefore, more effective if provided at multiple intervals prior toprocuring the organ. The amount of the induction agent in the dosedepends on (1) the induction agent or agents used, (2) the species, sex,and size of the donor, (3) the mode of administration of at least oneinduction agent, and (4) the frequency of dose administration.Typically, when the induction agent is administered over a series ofdoses, the dosage of induction agent may be less. One of skill in theart would be able to successfully determine how to manipulate thesedosing parameters in order to obtain in vivo induced hepatocyte culturesfor use in the method and bioreactor of the invention.

[0014] “Induction agent” means an agent that is capable of increasing orupregulating hepatocyte cell functions, particularly those enzymesinvolved with detoxification, particularly cytochrome P450 or theconjugative reactions involved in detoxification. It is also useful ifthe induction agent maintains or improves other hepatocyte cellfunctions including metabolic functions such as ammonia clearance andsynthetic functions such as albumin and transferring production.

[0015] Induction agents are selected from the group including but notlimited to: beta-naphthoflavone (BNF), phenobarbital,3-methylcholanthrene (3MC), ethanol, dexamethasone, arochlor 1254,2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), phenothiazine,chlorpromazine, isosafole, γ-chlordane, allylisopropylacetamide (AIA),trans-stilbene oxide, kepone, acetone, isoniazid, pyridine, pyrazole,4-methylpyrazole, pregnenolone 16α:-carbonitrile (PCN), troleandomycin(TAO), clotrimazole, clofibrate, clobuzarit, di(2-ethylhexyl)phthalate(DEHP), or mono-(2-ethylhexyl)phthalate (MEHP). It should be noted thatthe aforementioned terms in parentheticals are abbreviations known inthe art for the chemical names that precede them. The most preferredinduction agents of the group are: beta-naphthoflavone, phenobarbital,and 3-methylcholanthrene. In the most preferred method, the inductionagents are administered to a donor by injection to the intraperitonealarea. It should be noted that dosages recited herein are in terms ofmilligrams of induction agent per kilogram of donor bodyweight.Phenobarbital is administered preferably up to about 125 mg/kg, morepreferably between about 40 to about 80 mg/kg. Beta-naphthoflavone isadministered preferably up to about 180 mg/kg, more preferably betweenabout 10 to about 15 mg/kg. 3-methylcholanthrene is preferablyadministered up to about 25 mg/kg, more preferably between about 5 toabout 10 mg/kg. Some chemicals that are either functionally orstructurally similar to those listed may be identified by one of skillin the art for practicing the invention. While not wishing to be boundby theory, many of the chemicals listed are customarily classifiedtogether in the same chemical classes with a number of other aromatic orbarbituate compounds and are able to upregulate functional metabolicactivity of hepatocytes. Carrier agents, adjunct agents, encapsulationmeans, or a combination thereof may also be added with the inductionagent in the dose to regulate uptake and absorption rates of inductionagent. Carriers may be aqueous, such as water or saline, and may bebuffered with phosphate, borate, or citrate, for example. Non-aqueouscarriers may also be used, such as dimethylsulfoxide (DMSO) or benzene.The induction agent may also be released from an encapsulation means.

[0016] One or more induction agents may be used in vivo to upregulatethe enzymatic activity of the hepatocytes prior to isolation. A singleinduction agent may be administered to a donor one or more times priorto isolation. Induction agents may be combined, meaning as a mixture or‘cocktail’ at the same time, or serially, meaning separately atdifferent times, when administered to upregulate a profile of targetenzymes. The amount of induction agent contained in the dose should beenough to induce the hepatocytes to increase their functional metabolicactivity but not so much as to be lethal to the liver organ or to thedonor. The time that the induction agent is provided to a donor shouldbe long enough to result in upregulation of enzymatic detoxificationactivity, preferably at least about 24 hours prior isolation.

[0017] In vivo induction initiates upregulation of several functionaldetoxification enzymes such as cytochrome P450 isozymes and conjugatingenzymes so that the hepatocytes, after isolation and incorporation in abioreactor, sustain measurable detoxification activity for about a week.Non-induced hepatocyte cultures are not upregulated to the levels ofactivity found in in vivo induced hepatocyte cultures and do not sustainsuch levels for as long, only about 3 or 4 days.

[0018] To date, much of the cytochrome P450 isozyme work has beenperformed on either rat or human hepatocytes and therefore many of theknown cytochrome P450 isozymes have been identified and named based onthe correlation between the induction agents and the isozymes theyupregulate. Extending that knowledge to porcine hepatocytes, however,one will find both similarities and differences between the inductionagent and isozyme activity. The induction agents have effect on theexpected isozyme or its species-specific homolog. In the preferredembodiment, hepatocytes are isolated from porcine liver so the inductionagent or agents used will have effect on the expected isozyme or itsporcine homolog.

[0019] Table 1 summarizes the induction activity of the most preferredinduction agents for use in the invention along with their targetisozymes, and the substrates that the isozymes convert. Inducedhepatocytes initially express increased P450 isozyme activity onalkoxyresorufin substrates, converting them to resorufin, at a levelhigher than that of noninduced hepatocytes. A preferred level oftargeted P450 isozyme activity increase of in vivo induced hepatocytesover non-induced hepatocytes is at least about two (2)-fold for use inthe bioreactor of the invention. Certain induction agents are chosen totarget and upregulate particular isozymes responsible for conversion ofalkoxyresorufin substrates that may concomitantly upregulate conversionactivity on other substrates. This upregulation may occur by the same ordifferent pathways.

[0020] In the cytochrome P450 pathway, in vivo induction of a donorusing phenobarbital upregulates CYPIIB1 and CYPIIB2 isozymatic activityof hepatocytes, or the activity of their porcine homologs, onbenzyloxyresorufin (BROD) and pentoxyresorufin (PROD) substrates,respectively. Beta-naphthoflavone is specific for upregulation of CYPIA2and CYPIA1 isozymatic activity, or the activity of their porcinehomologs, on methoxyresorufin (MROD) and ethoxyresorufin (EROD)substrates, respectively. Methylcholanthrene upregulates CYPIIB1isozymatic activity, or its porcine homolog, to PROD; CYPIA2 isozymaticactivity, or its porcine homolog, on MROD; and CYPIA1 isozymaticactivity, or its porcine homolog, on EROD. Another widely used substrateto assess hepatic enzymatic activity is 7-ethoxycoumarin (7-EC). Thissubstrate is O-deethylated to yield a fluorescent product and is alsoindicative of oxidative metabolism of the cytochrome P450 enzymes. Theresults from these assays suggest that increases in isozymatic functionare obtained following in vivo induction. Furthermore, HPLC analysis ofthe detoxification processes in the liver show that drugs, such aslidocaine and diazepam, which are metabolized in the liver, are clearedat a much greater rate than in the noninduced state. This finding isclinically significant as drug overdoses are a major cause of hepaticfailure. TABLE 1 Summary of Induction Activity Conferred onAlkoxyresorufin Substrates by Hepatocytes Induced In Vivo Target Degreeof isozyme Isozyme or activity Porcine increase over Homolog noninducedInduction agent Substrates Thereto control Phenobarbital BROD CYPIIB2 20to 100-fold (40 to 80 mg/kg) PROD CYPIIB1  2 to 40-fold 7-EC CYPIA2 20to 50-fold Lidocaine CYPIA2 10 to 20-fold Diazepam CYPIIB1 20 to 50-foldBeta-Naphthoflavone MROD CYPIA2  2 to 10-fold (10 to 15 mg/kg) ERODCYPIA1  2 to 10-fold 3-Methylcholanthrene PROD CYPIIB1  2 to 10-fold (10to 15 mg/kg) MROD CYPIA2  2 to 10-fold EROD CYPIA1 10 to 20-foldDiazepam CYPIIB1  2 to 10-fold

[0021] The conjugation reaction pathway is another induction pathway forincreased conversion activity by hepatocytes. There are several knownconjugation reactions that may be upregulated by in vivo inductionmethods, such as the glucoronidation and sulfation conjugation reactionpathways. Glucuronidation is a primary mechanism for producing polarmetabolites of xenobiotics for excretion. Phenobarbital is involved withnot only cytochrome P450 isozyme activity but also conjugation enzymes.Alcohol, phenol, N-hydroxylamine, and carboxyl groups undergoO-glucoronidation; alkylamine, arylamine, amide, sulfonamide, andtertiary amine groups undergo N-glucoronidation; sulfhydryl groupsundergo S-glucoronidation; and tetrahydrocannabinol groups undergoC-glucoronidation. Enzymatic glucuronidation is accomplished by theenzyme UDP-glucuronyltransferase. Another conjugation pathway for thereduction of foreign compounds and drugs bearing a hydroxyl group issulfation. The class of sulfotransferase enzymes that may be upregulatedby in vivo induction include alcohol sulfotransferase, amineN-sulfotransferase, and phenol sulfotransferase.

[0022] If a recipient patient is in need of liver assist treatment foran indication where the expression of detoxification enzyme activity islow, a liver assist device may be prepared using a mixture of cellisolates having a profile of hepatocytes with a number of enzymeactivities upregulated to achieve the greatest range of detoxificationactivity and provide a tailor-made culture for treatment of acutefailure.

[0023] After the induction stage, the cells are isolated using amodification of the Seglen method as described in Seglen, P O.Preparation of isolated rat liver cells. In Methods in Cell Biology (D MPrescott, ed.) vol. 13. Academic Press (NY, N.Y.), 1976, incorporatedherein. The animal is anesthetized, opened, and the exposed liver iscannulated and perfused in situ with cold lactated Ringers solutionbefore excision to rinse blood and excess induction agent from the livertissue. The excised liver is transported to a biological safety cabinetwhere the remainder of the procedure may be performed under asepticconditions. The extracellular matrix that provides the physicalstructure of the liver is then digested by quickly perfusing the organwith warmed EDTA, preferably at 37° C., followed by perfusion of 1 mg/mlcollagenase at 37° C. until digestion appears complete (mean digestiontime is about 22 minutes). Further digestion is then stopped with theaddition of cold Hank's Balanced Salt Solution (HBSS) supplemented withcalf serum. Digestion of liver matrix releases cells and cell aggregatesfrom the matrix structure to create a suspension of cells. Undigestedtissue and gallbladder are excised and the remainder of the tissue ispassed through 200 micron and 100 micron stainless steel sieves torelease cells and cell aggregates. The cell suspension is washed twiceby centrifugation and decanting of rinse media and the cell pelletresuspended in media preferably after the third rinse. At this point,cells may be cultured in culture medium or cryopreserved in acryopreservation medium for long-term storage for future use.

[0024] The cells are cultured as a cell suspension or plated on asurface suitable for animal cell or tissue culture, such as a culturedish, flask, or roller-bottle, which allows for hepatocyte culture andmaintenance. The cells may be incorporated in a bioreactor, either insuspension or plated on a culture substrate such as a culture bead orfiber, or on a flat surface or membrane. Suitable cell growth substrateson which the cells can be grown can be any biologically compatiblematerial to which the cells can adhere and provide an anchoring meansfor the cell-matrix construct to form. Materials such as glass;stainless steel; polymers, including polycarbonate, polystyrene,polyvinyl chloride, polyvinylidene, polydimethylsiloxane,fluoropolymers, and fluorinated ethylene propylene; and siliconsubstrates, including fused silica, polysilicon, or silicon crystals maybe used as a cell growth surfaces. To enhance cell attachment orfunction, or both, the cell growth surface material may be chemicallytreated or modified, electrostatically charged, or coated withbiologicals such as with extracellular matrix components or peptides. Inone embodiment, the hepatocytes are cultured either within or on thesurface of extracellular matrix disposed on the culture surface such ascollagen in the form of a coating or a gel. In another embodiment, thehepatocytes are cultured on either a liquid-permeable membrane or agas-permeable membrane. Other cells present in liver may also beincluded with the induced hepatocytes such as endothelial cells; Kupfercells, a specialized macrophage-like cell; and, fibroblasts. Aco-culture of hepatocytes with one or more of these or other types ofcells may be desirable to optimize hepatocyte functioning.

[0025] The in vivo induced hepatocytes are preferably seeded in abioreactor that is used as, or is incorporated into a LAD. Some LADdesigns are based on a hollow fiber cartridge design where thehepatocytes are seeded either in the lumen of the hollow fibers or onthe outside of the hollow fibers. The hollow fiber serves as a culturesubstrate that allows for liquid or gas transport across the hollowfiber. Other LAD designs incorporate a flat planar culture substrate.Hepatocyte culture between two collagen gel layers is described in U.S.Pat. Nos. 5,602,026, and 5,942,436 to Dunn, et al. Another design usinga planar culture substrate is disclosed in U.S. Pat. No. 5,658,797 andin International PCT Publication No. WO 96/34087 to Bader, et al. Someflat planar substrates may be micropatterned so that two or more celltypes may be cultured together, as a co-culture, in discrete regions ona substrate such as those described by Bhatia, et al. The disclosures ofthese aforementioned patents that disclose culture substrates andmethods and their use as a bioreactor device to treat patients in needof liver assist are incorporated herein by reference. A preferredbioreactor design for the culture of hepatocytes incorporates agas-permeable, liquid impermeable membrane that defines two regions of abioreactor chamber. Hepatocytes are seeded on the surface of themembrane cultured in a liquid medium while engaging in oxygenation andother gas transfer not only in the culture medium but also across themembrane. In alternative embodiments, the membrane is treated to improvecell adhesion such as by modifying the electrical charge of themembrane, as by corona discharge, or by treating or coating the membranewith extracellular matrix components, peptides, cell-adhesion moleculesor other chemicals. A preferred coating for the membrane is collagen.

[0026] When cultured, the cells are preferably contacted with a cellculture medium for a time to maintain their metabolic activity andoptimal hepatocyte function. Albeit in varying concentrations, cellculture media provide a basic nutrient source for cells in the form ofglucose, amino acids, vitamins, and inorganic ions, together with otherbasic media components. Culture media generally comprises a nutrientbase further supplemented with one or more additional components such asamino acids, growth factors, hormones, anti-bacterial agents andanti-fungal agents. One preferred medium for use in the method afterhepatocyte isolation comprises: Williams' E medium, newborn calf serum(NBCS), glucose, insulin, glucagon, hydrocortisone, HEPES, epidermalgrowth factor (EGF), and glutamine. In a more preferred embodiment, theculture medium comprises: Williams' E media supplemented with up to 1%newborn calf serum (NBCS), 4.5 g/l glucose, 0.5 U/ml insulin, 7 ng/mlglucagon, 7.5 μg/ml hydrocortisone, 10 mM HEPES, 20 ng/ml EGF, and 200mM glutamine. Other concentrations for the aforementioned mediumcomponents or their functional equivalents may be determined for use byone of skill in the art of hepatocyte culture.

[0027] In an alternate preferred embodiment, hepatocytes arecryopreserved for storage after isolation until needed for incorporationin a bioreactor. Cryopreservation of cell suspensions, cell monolayers,and engineered tissue constructs are known in the art ofcryopreservation. Cryopreservation is useful for long term storage,banking, and shipping. When needed, the cultures are removed from frozenstorage, thawed, rinsed of cryopreservative, and ready for use.

[0028] After either isolation or removal from cryopreservation storage,the in vivo induced hepatocytes are preferably incorporated and culturedin a bioreactor. Hepatocytes from a single isolation induced with eithera single or multiple doses of the same induction agent, or a number ofinduction agents, may be used. In one alternative embodiment,hepatocytes isolated from a non-induced donor are cultured in abioreactor with hepatocytes isolated from an in vivo induced donor. Inanother alternative embodiment, hepatocytes from two or more donorisolations induced by the same induction agent or at least two differentinduction agents are combined together in the same bioreactor. If thebioreactor has multiple culture chambers or regions, hepatocytes fromdifferent donors that have been pre-treated with different inductionagents may be segregated but used together for the overall functioningof the bioreactor. Combining in vivo induced hepatocyte cultures thathave different enzyme activity profiles in a bioreactor used as a LADwill benefit a patient treated with the cultures in the bioreactor. Inone embodiment, the bioreactor may contain several isolations ofdifferent in vivo induced hepatocyte cultures to provide the patientwith a full profile of upregulated enzymes to achieve the greatest rangeof detoxification activity. An alternative embodiment is one where thepatient may be treated with a bioreactor seeded with one or moreisolations of in vivo induced hepatocytes with certain selectedenzymatic activities that augment or replace certain levels of enzymaticactivity where the patient's liver expresses low levels of a certaindetoxification enzyme.

[0029] The bioreactor may be used to culture the cells to produce cellproducts or to functionally act on substances, such as toxins normallymetabolized by liver. The bioreactor may serve as, or be an integralpart of, a liver assist device to treat a patient in need of liverassist. Hepatocytes having upregulated enzymatic activity may be used invarious types of bioreactors used as liver assist devices. Bioreactorssuited for this purpose comprise suspension means, hollow fibers, radialflow surfaces and planar substrates as cell culture.

[0030] Hepatocytes that have been induced in vivo are useful to treat apatient in need of liver assist when cultured in a bioreactor that isused as, or is incorporated into, a liver assist device. Usually,hepatocyte perfusion medium and the patient's plasma or blood arecirculated through the device in separate flow loops. The flow loopscontact each other via a membrane for the exchange of gases, toxins, andalbumin but also provide an immunological barrier between thehepatocytes and the patient.

[0031] The following examples are provided to better explain thepractice of the present invention and should not be interpreted in anyway to limit the scope of the present invention. Those skilled in theart will recognize that various modifications can be made to the methodsdescribed herein while not departing from the spirit and scope of thepresent invention.

EXAMPLES Example 1

[0032] In Vivo Induction and Isolation of Hepatocytes

[0033] A series of in vivo induction studies utilized various doses andinjection regimes ranging from 40 to 80 mg/kg on days −4 to −1 withphenobarbital in PBS; 3-methlycholanthrene (in DMSO or benzene);or,β-naphthoflavone (in DMSO) given at 5 to 15 mg/kg on days −3, −2 and/or−1 prior to surgical removal of the liver. A summary of various trialsis presented in Table 2. Yorkshire/Hampshire crossbred pigs weighing8.3±3.0 kg were obtained from E M Parsons (Hadley, Mass.). Allinjections of induction agents were administered into the peritonealcavity. TABLE 2 In vivo Induction Agents, Dosages, and Frequency DosageDosing (per kg frequency donor (days prior Induction agent/ body- toliver Trial Carrier agent weight) procurement) Trial 1 Phenobarbital/PBS80 mg/kg Days -3 and -2 Trial 2 Phenobarbital/PBS 40 mg/kg Days -3, -2and -1 Trial 3 Phenobarbital/PBS and 40 mg/kg Days -3, -2 and -1β-Naphthoflavone/DMSO 10 mg/kg Days -3, -2 and -1 Trial 4Phenobarbital/PBS and 40 mg/kg Days -4, -3 and -2 β-Naphthoflavone/DMSO10 mg/kg Days -4, -3 and -2 Trial 5 3-methlycholanthrene/DMSO  5 mg/kgDays -3, -2 and -1 Trial 6 3-methlycholanthrene/benzene 10 mg/kg Days-3, -2 and -1 Trial 7 3-methlycholanthrene/benzene 10 mg/kg Day -1 onlyTrial 8 β-Naphthoflavone/DMSO 15 mg/kg Day -1 only

[0034] Heparin (Elkins-Sinn, Cherry Hill, N.J.) was administeredintravenously at 0.5 mg/kg and donors were anesthetized with a mixtureof Telazol (7-10 mg/kg, Fort Dodge Laboratories, Fort Dodge, Iowa) andRompun (5 mg/kg, Miles, Inc., Shawnee Mission, Kans.). Plane ofanesthesia was maintained with isoflurane gas and all procedures wereperformed in compliance with ACUC guidelines.

[0035] Cells were isolated using a modification of the Seglen methodwhich has been described earlier (Seglen, P., Preparation of isolatedrat liver cells, In Meth. in Cell. Bio. (D M Prescott, ed.), vol. 13.Academic Press (NY, N.Y.), 1976.). Briefly, the exposed liver wascannulated and perfused in situ with cold Lactated Ringers (Baxter,Deerfield, Ill.) at 250 ml/minute before excision and transport to thelab. The liver was quickly warmed and perfused with 0.2% EDTA at 37° C.This was followed by perfusion of 1 mg/ml collagenase (LifeTechnologies, Grand Island, N.Y.) at 37° C. until digestion appearedcomplete (mean digestion 22±4 min). Further digestion was stopped withthe addition of cold HBSS (BioWhittaker, Walkersville, Md.) supplementedwith 10% NBCS (Hyclone, Logan, Utah). Undigested tissue and gall bladderwere excised and the remainder of the tissue passed through 200 and 100micron stainless steel sieves (Fisher Scientific, Pittsburgh, Pa.). Thecell suspension was washed twice and the cell pellet resuspended inculture media. Viability was determined by Trypan blue exclusion andCalcein AM staining (Molecular Probes, Eugene, Oreg.). Hepatocytesderived from the isolation process following in vivo induction showlower viability when compared to control conditions (77% n=7 versus 89%n=40), yet the cells detoxify xenobiotics at a greater rate. Immediatelyafter isolation, 10⁶ cells were assayed for total protein with the BCAkit (Pierce Biochemical, Rockford, Ill.).

[0036] Cells were plated onto 60 mm tissue culture dishes at a densityof 2×10⁶ cells per dish (Corning, Corning, N.Y.) in Williams' E mediasupplemented with 1% NBCS (newborn calf serum), 4.5 g/L glucose, 0.5U/ml bovine insulin, 7 ng/ml glucagon, 7.5 μg/ml hydrocortisone, 10 mMHEPES (Sigma), 20 ng/ml EGF, 200 mM glutamine (Life Technologies), 10IU/ml penicillin and 10 μg/ml streptomycin (BioWhittaker). Hepatocyteswere incubated at 37° C. in humidified 10% CO₂. Culture medium waschanged on day 1 post-isolation and then every 2-3 days. Media samplestaken at each change were stored for assay of albumin and urea.

[0037] For comparison, in vitro induced hepatocyte cultures wereprepared. Hepatocytes were procured using the method described above butfrom non-induced pigs and then induced in culture. Cells were platedonto 60 mm tissue culture dishes (Corning, Corning, N.Y.) in Williams' Emedia supplemented with 1% NBCS (newborn calf serum), 4.5 g/L glucose,0.5 U/ml bovine insulin, 7 ng/ml glucagon, 7.5 μg/ml hydrocortisone, 10mM HEPES (Sigma), 20 ng/ml EGF, 200 mM glutamine (Life Technologies), 10IU/ml penicillin and 10 μ/ml streptomycin (BioWhittaker). Experimentalconditions were treated with culture medium containing any one of thefollowing: 2 mM phenobarbital (PB; Sigma) for 96 hours, 50 μMβ-naphthoflavone (BNF; Sigma) for 24 hours, or 5 μM 3-methlycholanthrene(3-MC; Sigma) for 24 hours.

Example 2

[0038] Cytochrome P450 Function Measured in Hepatocytes

[0039] The dealkylation of phenoxazone substrates has provided apowerful tool for investigating cytochrome P450 activities and isozymeprofiles. Specifically, the dealkylation of the phenoxazone ethersethoxyresorufin (EROD), methoxyresorufin (MROD), benzyloxyresorufin(BROD) and pentoxyresorufin (PROD) have allowed researchers to study theeffects of induction agents on individual isozymes, namely, CYPIA1,CYPIA2, CYPIIB1 and CYPIIB2, respectively.

[0040] To measure increased isozyme activity in both in vivo and invitro induced cultures compared to non-induced controls, both in vivoinduced and in vitro induced cultures from Example 1 were incubated withEROD, MROD, PROD or BROD (Molecular Probes) at 5 μM (in Williams' Emedia without serum and phenol red). Dicumarol (80 μM, Sigma) wasincluded in the incubation to limit cytosolic degradation of theresorufin end product. Samples were analyzed in a Turner 450 fluorometerat 540_(ex) and 585_(em) nm. Resorufin formation was quantified using astandard curve that was linear from 0.5 to 130 nM and data are expressedas net resorufin gain over background fluorescence. In this assay,conversion of the alkoxyresorufins to resorufin corresponds to anincrease in fluorescence such that the ratio of fluorescences, oractivity, corresponds directly to the level of P450 activity of aparticular set of isoenzymes.

[0041] Phenobarbital induction led to increased expression of porcineCYPIIB 1 and CYPIIB2 in both in vitro and in vivo induced hepatocytecultures, however, the level of cytochrome P450 isozyme activity of invivo induced cultures was much greater. In FIG. 1, the effects ofphenobarbital (referred to as “phenobarb” and “PB” in the Figure) on invitro and in vivo induced hepatocytes are graphically presented. InFIGS. 1a and 1 b, the effect of phenobarbital was observed oncytochromes CYPIIB1 and CYPIIB2. Two million hepatocytes, which wereexposed to phenobarbital for 96 hours in vitro, were incubated with thesubstrates for 3 hours and media samples collected for analysis offluorescence. CYPIIB2 shows an 8.4-fold increase in function overnoninduced controls following 96 hours after in vitro induction. At thispoint in culture, the noninduced cells show a low level of functiontypically seen on day 4 of culture. CYPIIB 1 isozyme metabolizes PROD ata 3-fold higher rate following phenobarbital treatment. This lower levelof induction with CYPIIB1 isozyme observed in porcine hepatocytes isalso seen with the rat model where CYPIIB2 shows a greater level ofinducibility (typically, 20-30-fold versus 5 to 10-fold (Thomas, P. E.,Reik, L. M., Ryan, D. E., and Levin, W., 1983, Induction of twoimmunochemically related rat liver cytochrome P-450 isozymes,cytochromes P-450c and P-450d, by structurally diverse xenobiotics, JBiol Chem 258:4590-4598.). The impact of three daily injections, invivo, of phenobarbital to the donor prior to that harvest of the liveris seen in FIG.s 1 c and 1 d. A dramatic increase in CYPIIB2 and CYPIIB1was observed in vivo, substantially larger than what was seen in the invitro induction effect. A 70-fold increase in function was obtainedfollowing in vivo induction for CYPIIB2, while a 38-fold upregulation isrecorded with CYPIIB1. These data were collected on day one of culturefrom the standard plating density of 2×10⁶ on the 60 mm TC dish. Therate of resorufin formation from the BROD substrate was approximately10.4 nM resorufin per minute from the in vivo induction (day 1). Thiscompared favorably with the in vitro induction rate of about 0.33 nMresorufin formed per minute (day 4), 69-times higher than the averagecontrol rate of resorufin formed on day 1 from BROD at 0.15 nM perminute.

[0042] In vivo induction with phenobarbital also demonstratedmaintenance of hepatocyte function in induced hepatocyte cultures longerthan non-induced cultures. FIG. 2 shows the measurable enzyme activityat four days after plating of hepatocytes induced in vivo with 40 to 80milligrams of phenobarbital/PBS per kilogram donor bodyweight once eachday, 96 hours (4 doses) prior to liver harvest. Four days after plating,the cytochrome P450 activity of in vivo-induced hepatocytes was reducedby about 50% but were still highly active compared to noninducedcontrols.

[0043] β-Naphthoflavone, both in vitro and in vivo, upregulates CYPIA1in porcine hepatocytes. In vitro induction of porcine CYPIA1 (ERODSubstrate) following 24 hours of induction in media containing 50 μMβ-Naphthoflavone (“BNF”) caused a 14.8-fold increase in function overcontrol metabolic conversion of EROD. The in vitro induction increasesthe resorufin formed per minute from control levels of 0.27 nM to 4.0nM.

[0044]FIG. 3 depicts the effects of 3-methlycholanthrene (“3 MC” or“MC”) on both in vitro and in vivo induced hepatocyte cultures. In vitroinduction of CYPIA2 (MROD), FIG. 3a, and CYPIA1 (EROD), FIG. 3b, isshown. Porcine hepatocytes were cultured in media containing 5 μM3-methylcholanthrene for 24 hours. Both CYPIA1 and CYPIA2 were affected,increasing their conversion rates to 18-fold and 4-fold that of thecontrol rates, respectively. These data were also for two millionhepatocytes on day 2 of culture. In vivo induction of CYPIIB1 (PROD),FIG. 3c, CYPIA2 (MROD), FIG. 3d, and CYPIA1 (EROD), FIG. 3e, by3-methlycholanthrene are also shown. FIGS. 3c, d, and e demonstrate thewider range of impact that 3-methlycholanthrene possessed in vivo.CYPIA1 and CYPIA2 were affected by 3-methylcholanthrene, 24-fold and7.6-fold over noninduced controls, respectively. An 8.4-foldupregulation of CYPIIB1 (PROD) was measured following in vivoadministration of the induction agent. This effect on CYPIIB1 was notobserved following the in vitro induction with the same induction agent.

Example 3

[0045] Lidocaine and Diazepam Clearance

[0046] Lidocaine (Paddock Laboratories Inc., Minneapolis, Minn.)clearance was assayed using a modification of the protocol of Nyberg etal. Pharmacokinetic analysis verifies P450function in in vitro and invivo application of a bioartificial liver, ASAIO, 39:M252-M256, 1993.Lidocaine (740 μM) was added to the control, and both in vivo inducedand in vitro induced cultures of Example 1 for the indicated times;media samples were then collected and frozen until extraction. Solidphase extraction was performed with Oasis cartridges (Waters Corp.,Milford, Mass.) and a Waters extraction manifold as follows: Cartridgeswere primed with 99% MeOH 1% HCl and 0.5 M Borax. The sample was loadedonto the column, washed with 0.5 M Borax, eluted with MeOH/HCl and thenevaporated and reconstituted with 250 μl of mobile phase (85% 50 mMN₄HPO₄+10 mM Hexanesulfonic Acid, pH 3.0, 15% Acetonitrile). The reversephase HPLC was carried out with a flow rate of 1 ml/minute on aMicrosorb C8 column (Rainin Instrument Co., Woburn, Mass.) at roomtemperature and monitored at 214 nm. Lidocaine eluted at approximately37 minutes; MEGX, the major metabolite, eluted at 27 minutes. In thisassay, high percentages of lidocaine cleared correspond to high P450activity; the higher these percentages, the greater the P450 activity.

[0047] Diazepam clearance was assayed in the cultures of Example 1 usinga method similar to that of Jauregui et al. Xenobiotic induction ofP-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenaseactivities in primary cultures of adult rat hepatocytes. Xeno,21(9):1091-106. 1991. After addition of 70 μM diazepam (Sigma) for 48hours, media samples were collected and frozen until assay. Oasis solidphase extraction was performed on each sample with a priming step of100% MeOH, followed by RODI. Samples were loaded onto the column andwashed with 5% MeOH in RODI. Elution off the column was achieved with100% MeOH. As with lidocaine, the samples were evaporated andreconstituted with mobile phase (65% MeOH, 35% 0.01 M Ammonium Acetate,pH 6.0). This reverse phase HPLC run was conducted at a flow rate of 1.0ml/min through a micro-Bondpak C18 column (Waters) with absorbance setat 254 nm. The temperature was held constant at 24.5° C. Diazepam elutedat approximately 11 minutes, with metabolites nordiazepam, temazepam andoxazepam eluting at 10, 8, and 7 minutes, respectively. In this assay,high percentages of initial diazepam cleared and converted tonordiazepam and temazepam correspond to high P450 activity. Similarly,high percentages of lidocaine cleared correspond to high P450 activity.The higher these percentages, the greater the P450 activity.

[0048] Results showed that phenobarbital induction can upregulateCYPIIB1 in the resorufin assay (PROD) (see Example 2) and here,similarly increased diazepam clearance rates. This assay, in particular,may be most clinically significant as it has been postulated thatbenzodiazepine-like compounds are implicated in human hepaticencephalopathy (Jones, E. A., Gammel, S. H., Basile, A. S., Mullen, K.D., Bassett, M. L. Schaffer, D. F., and Skolnick, P., 1989, Hepaticencephalopathy and benzodiazepine receptor ligands. In HepaticEncephalopathy: Pathophysiology and Treatment, ed. by R. F. Butterworthand G. P. Layrargues (Clifton: Humana Press), pp. 273-286).

[0049] Lidocaine clearance showed an upregulation following in vivophenobarbital induction to about 10 to 20-fold over non-induced controlcultures. Diazepam clearance showed an upregulation following in vivoinduction by 3-methlycholanthrene to about 2 to 10-fold over non-inducedcontrol cultures.

Example 4

[0050] 7-Ethoxycoumarin Metabolism

[0051] 7-Ethoxycoumarin (7EC, Sigma) metabolism was measured byincubating cells (control and both in vivo induced and in vitro inducedcells) of Example 1 at 37° C. with 375 μg/ml of 7-ethoxycoumarin inphenol red free culture media for the indicated time. Samples were thenanalyzed for the fluorescent product using a Turner 450 fluorometer withhalogen illumination at 360_(ex) and 415_(em).

[0052] 7-Ethoxycoumarin O-deethylation is also higher followinginduction with phenobarbital. The impact of in vivo phenobarbitaladministration is observed on 7-ethoxycoumarin metabolism, as shown inFIG. 4. In this experiment, 24 hours after adding the 7-ethoxycoumarinsubstrate, the induced hepatocytes showed a dramatic production of 7-OHcoumarin (umbelliferone), the major metabolite of 7-ethoxycoumarin. Theinduction increased umbelliferone production rates from 2.58±1.9 nM perhour to 346±41.4 nM per hour.

Example 5

[0053] Albumin and Transferring Measurement

[0054] Albumin secretion was measured using a standard competitive ELISAformat. Maxisorp Microtiter plates (Nunc) were coated overnight with 200μg/ml porcine albumin (Accurate Chemical, Westbury, N.Y.). Following awash step with Tween 20 (Pierce, Rockford, Ill.), 50 μl of sample orstandard (Accurate) was incubated with a HRP (horseradishperoxidase)-conjugated goat anti-pig albumin antibody (Bethyl Labs,Montgomery, Tex.) for 90 minutes. Color was produced by addition of OPDsubstrate (Pierce) and the reaction was stopped by adding H₂SO₄. Plateswere read at 490 nm using SoftMax Pro software and to a SpectraMax 250plate reader (Molecular Devices).

[0055] Transferring was similarly assessed using Maxisorp plates coatedovernight with 100 μg/ml swine transferring (Accurate). Following a washwith Tween 20/PBS, 50 μl of sample or standard was incubated withHRP-conjugated rat anti-swine transferring (Accurate) for 90 minutes.Color development was produced, stopped and analyzed as above.

[0056] Synthetic functions of the hepatocytes, namely albumin andtransferring production were maintained, indicating that the inductiontreatment did not downregulate these synthetic functions in isolatedporcine hepatocytes.

Example 6

[0057] Hepatocellular Deamination of Medium Based on Synthesis of Urea

[0058] The clearance of ammonia, its salts, and aminated components inthe medium, through deamination, is believed to be a critical functionof hepatocytes in vivo and a desired function of these cells as part ofa liver-assist device. Deamination results in the formation of urea,which in vivo is cleared by the renal system.

[0059] The synthesis of urea by in vivo induced hepatocytes weremeasured using a colorimetric method for the determination of nitrogen,available as Kit #640-B from Sigma Diagnostics (St. Louis, Miss.).Samples were collected periodically after seeding of cells into devicesand treated with urease to hydrolyze urea to NH₃ and CO₂. The resultingNH₃ then was reacted with hypochlorite and phenol in the presence of thecatalyst, sodium nitroprusside, to form indophenol. The opticalabsorbance of the resulting solution of indophenol was measured at 570nm and converted to concentration of urea in the original sample using astandard curve. Data were expressed as amount of urea produced perdevice per day by multiplying the concentrations by volume of medium inthe device and dividing by number of days since sampling. The synthesisof urea by in vivo induced hepatocytes was maintained indicating thatthe induction treatment did not downregulate this synthetic function inisolated porcine hepatocytes.

Example 7

[0060] In Vivo Induced Hepatocytes Cultured in a Bioreactor Device

[0061] To test the feasibility of in vivo induced cells for use in a newbioreactor design, incorporating new structural features and materialcomposition, prototype bioreactor device units were compared to standardtissue culture dishes. A bioreactor device was constructed having anassembly of upper and lower housings, separated by a gas-permeable,liquid-impermeable membrane to form upper and lower chambers. The upperchamber had an access window for addition of cells and media transferand a cover to allow aseptic transportation of the assembled deviceafter seeding with cells and to prevent spillage or exposure of theinterior of the device to contaminants. The membrane used was Polyflex®(Plastics Suppliers, Inc., Columbus, Ohio), a 0.002″-thick film ofpolystyrene that had been treated with a corona discharge. The upper andlower polycarbonate housings were bolted together to sandwich themembrane/frame assembly, and a gas and liquid-tight seal was formedbetween the housings using O-rings disposed between the housings. Allassembly steps, unless otherwise noted, were conducted in a biologicalsafety cabinet and occurred after sterilizing all parts by autoclavingor other proven treatment (e.g., gamma irradiation or exposure to anoxidizing gas such as ethylene oxide, peracetic acid, and/or hydrogenperoxide). All materials were handled with either sterile tweezers orgloves within the cabinet.

[0062] Phenobarbital in phosphate buffered saline was administered atabout 40 mg per kilogram of bodyweight at 4, 3, and 2 days to a porcinedonor, a Yorkshire/Hampshire crossbred pig weighing 8.3±3.0 kg, prior tosurgically removing the liver and isolating the hepatocytes according tothe Seglen method disclosed in Example 1.

[0063] Primary hepatocytes were suspended in complete medium (Williams Emedium supplemented with 4.5 g/L glucose, 0.5 U/mL bovine insulin, 7ng/mL glucagon, 7.5 μg/mL hydrocortisone, 10 mM HEPES, 20 ng/mL EGF, 20mM glutamine, 10 IU penicillin, and 10 μg streptomycin) with 1% new-borncalf serum (NBCS) were obtained from porcine donors with the followingprocedure.

[0064] Before seeding cells, the membranes of the bioreactor devices andthe tissue culture dishes were pre-coated with a sterile 4 mL volumesolution of 40 μg/mL Type I collagen in water for 45 minutes, followedby aspiration of this solution and rinsing with an equal volume ofsterile phosphate-buffered saline (PBS), prior to seeding of cells.

[0065] A suspension of cells in medium was evenly suspended and wereseeded at an initial density of 2×10⁶ cells per device. The cover to thedevice was removed, the cell suspension pipetted onto the membrane, thedevice agitated carefully to distribute the liquid evenly onto thesurface of the membrane, and the cover replaced. The cell-seeded devicesand tissue culture dishes were transferred to an incubator at 37° C. and85% relative humidity in 10% CO₂.

[0066] After approximately 18-24 hours the device was removed from theincubator, placed back in the biological safety cabinet, cover removed,and the medium aspirated using a sterile Pasteur pipette. The isozymeactivity of the hepatocytes in both the tissue culture plates and theprototype device were assayed according to the methods of Example 2.

[0067] BROD conversion (CYPIIB2) and EROD conversion (CYPIA1) weresimilar on both the tissue culture and bioreactor device conditions.These results indicate that in vivo induced hepatocytes havingupregulated enzymatic detoxification activity may be used in abioreactor.

[0068] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be obvious to one of skill in the art thatcertain changes and modifications may be practiced within the scope ofthe appended claims.

We claim:
 1. A method for increasing detoxification enzyme activity ofone or more hepatocyte isolated from a donor liver, comprising:administering at least one induction agent to a non-human donor in anamount sufficient to increase detoxification enzyme activity of one ormore hepatocyte, isolating the hepatocyte from the donor liver,incorporating the isolated hepatocyte in a bioreactor, wherein theinduction agent is selected from the group consisting of:beta-naphthoflavone, phenobarbital, 3-methylcholanthrene, ethanol,dexamethasone, arochlor 1254, 2,3,7,8-tetrachlorodibenzo-p-dioxin,phenothiazine, chlorpromazine, isosafole, γ-chlordane,allylisopropylacetamide, trans-stilbene oxide, kepone, acetone,isoniazid, pyridine, pyrazole, 4-methylpyrazole, pregnenolone16α-carbonitrile, troleandomycin, clotrimazole, clofibrate, clobuzarit,di(2-ethylhexyl)phthalate, and mono-(2-ethylhexyl)phthalate.
 2. Themethod of claim 1, wherein the non-human donor is a mammalian donor. 3.The method of claim 2, wherein the mammalian donor is a porcine donor.4. The method of claim 1, wherein phenobarbital is administered up toabout 125 mg/kg of donor bodyweight.
 5. The method of claim 1, whereinphenobarbital is administered between about 40 to about 80 mg/kg ofdonor bodyweight.
 6. The method of claim 1, wherein beta-naphthoflavoneis administered up to about 180 mg/kg of donor bodyweight.
 7. The methodof claim 1, wherein beta-naphthoflavone is administered between about 10to about 15 mg/kg of donor bodyweight.
 8. The method of claim 1, wherein3-methylcholanthrene is administered up to about 25 mg/kg of donorbodyweight.
 9. The method of claim 1, wherein 3-methylcholanthrene isadministered between about 5 to about 10 mg/kg of donor bodyweight. 10.The method of claim 1, wherein the induction agent is administered tothe porcine donor intraperitoneally.
 11. The method of claim 1, whereinthe induction agent is administered at multiple intervals as separatedoses.
 12. The method of claim 1, wherein the induction agent isadministered at one time as a single dose.
 13. The method of claim 1,wherein the induction agent is administered serially.
 14. The method ofclaim 1, wherein two or more induction agents are administered to thedonor.
 15. The method of claim 14, wherein the induction agents areadministered at multiple intervals as separate doses.
 16. The method ofclaim 14, wherein the induction agents are administered at one time as asingle dose.
 17. The method of claim 14, wherein the induction agentsare administered serially.
 18. The method of claim 1, comprising two ormore hepatocyte cell cultures wherein at least one hepatocyte cellculture is isolated from a donor that had been administered theinduction agent in an amount sufficient to increase detoxificationactivity of the hepatocytes.
 19. The method of claim 1, comprising, twoor more hepatocyte cell cultures wherein each hepatocyte cell culture isisolated from a different donor, and wherein the different donors wereadministered different induction agents in an amount sufficient toincrease detoxification activity of the hepatocytes.
 20. The method ofclaim 1, wherein the induction agent is phenobarbital and wherein thethus induced hepatocytes have a functional cytochrome P450 isozymeactivity on BROD substrates which is about 20 to about 100-fold greaterthan hepatocytes isolated from a mammalian donor that was notadministered an induction agent.
 21. The method of claim 1, wherein theinduction agent is phenobarbital and wherein the thus inducedhepatocytes have a functional cytochrome P450 isozyme activity on PRODsubstrates which is about 2 to about 40-fold greater than hepatocytesisolated from a mammalian donor that was not administered an inductionagent.
 22. The method of claim 1, wherein the induction agent isphenobarbital and wherein the thus induced hepatocytes have a functionalcytochrome P450 isozyme activity on 7-ethoxycoumarin substrates which isabout 20 to about 50-fold greater than hepatocytes isolated from amammalian donor that was not administered an induction agent.
 23. Themethod of claim 1, wherein the induction agent is phenobarbital andwherein the thus induced hepatocytes have a functional cytochrome P450isozyme activity on lidocaine which is about 10 to about 20-fold greaterthan hepatocytes isolated from a mammalian donor that was notadministered an induction agent.
 24. The method of claim 1, wherein theinduction agent is phenobarbital and wherein the thus inducedhepatocytes have a functional cytochrome P450 isozyme activity onlidocaine which is about 20 to about 50-fold greater than hepatocytesisolated from a mammalian donor that was not administered an inductionagent.
 25. The method of claim 1, wherein the induction agent isbeta-naphthoflavone and wherein the thus induced hepatocytes have afunctional cytochrome P450 isozyme activity on MROD substrates which isabout 2 to about 10-fold greater than hepatocytes isolated from amammalian donor that was not administered an induction agent.
 26. Themethod of claim 1, wherein the induction agent is beta-naphthoflavoneand wherein the thus induced hepatocytes have a functional cytochromeP450 isozyme activity on EROD substrates which is about 2 to about10-fold greater than hepatocytes isolated from a mammalian donor thatwas not administered an induction agent.
 27. The method of claim 1,wherein the induction agent is 3-methylcholanthrene and wherein the thusinduced hepatocytes have a functional cytochrome P450 isozyme activityon PROD substrates which is about 2 to about 10-fold greater thanhepatocytes isolated from a mammalian donor that was not administered aninduction agent.
 28. The method of claim 1, wherein the induction agentis 3-methylcholanthrene and wherein the thus induced hepatocytes have afunctional cytochrome P450 isozyme activity on MROD substrates which isabout 2 to about 10-fold greater than hepatocytes isolated from amammalian donor that was not administered an induction agent.
 29. Themethod of claim 1, wherein the induction agent is 3-methylcholanthreneand wherein the thus induced hepatocytes have a functional cytochromeP450 isozyme activity on EROD substrates which is about 10 to about20-fold greater than hepatocytes isolated from a mammalian donor thatwas not administered an induction agent.
 30. The method of claim 1,wherein the induction agent is 3-methylcholanthrene and wherein the thusinduced hepatocytes have a functional cytochrome P450 isozyme activityon diazepam substrates which is about 2 to about 10-fold greater thanhepatocytes isolated from a mammalian donor that was not administered aninduction agent.
 31. The method of claim 1, wherein the bioreactor isfor use in a liver assist device.
 32. The method of claim 1, wherein thebioreactor is a liver assist device.